The chemical modification of alpha-chymotrypsin with both hydrophobic and hydrophilic compounds stabilizes the enzyme against denaturation in water-organic media.

We considered alpha-chymotrypsin (CT) in homogeneous water-organic media as a model system to examine the influence of enzyme chemical modification with hydrophilic and hydrophobic substances on its stability, activity and structure. Both types of modifying agents may lead to considerable stabilization of the enzyme in water-ethanol and water-DMF mixtures: (i) the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value is broadened and (ii) the range of organic cosolvent concentration at which the residual enzyme activity is observed is increased. We found that for both types of modification the stabilization effect can be correlated with the changes in protein surface hydrophobicity/hydrophilicity brought about by the modification. Circular dichroism studies indicated that the effects of these two types of modification on CT structure and its behavior in water-ethanol mixtures are different. Differential scanning calorimetry studies revealed that after modification two or three fractions or domains, differing in their stability, can be resolved. The least stable fractions (or domains) have properties similar to native CT.     

Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain.

Membrane type (MT) matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn(2+)- and Ca(2+)-dependent MMPs. To investigate the proteolytic capabilities of human MT4-MMP (i.e. MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies in Escherichia coli, was purified to homogeneity and refolded in the presence of Zn(2+) and Ca(2+). While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-[2-mercapto-4-methylpentanoyl]-LG-OEt and Mca-PLGL-Dpa-AR-NH(2) with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-alpha converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH(2). Catalytic efficiency with the pro-tumor necrosis factor-alpha converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(a3) = 6.2, pK(a2) = 8.3, and pK(a1) = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn(2+)-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A.     

Characterization of the dimerization interface of membrane type 4 (MT4)-matrix metalloproteinase

MT4-MMP (MMP17) belongs to a unique subset of membrane type-matrix metalloproteinases that are anchored to the cell surface via a glycosylphosphatidylinositol moiety. However, little is known about its biochemical properties. Here, we report that MT4-MMP is displayed on the cell surface as a mixed population of monomeric, dimeric, and oligomeric forms. Sucrose gradient fractionation demonstrated that these forms of MT4-MMP are all present in lipid rafts. Mutational and computational analyses revealed that Cys(564), which is present within the stem region, mediates MT4-MMP homodimerization by forming a disulfide bond. Substitution of Cys(564) results in a more rapid MT4-MMP turnover, when compared with the wild-type enzyme, consistent with a role for dimerization in protein stability. Expression of MT4-MMP in Madin-Darby canine kidney cells enhanced cell migration and invasion of Matrigel, a process that requires catalytic activity. However, a serine substitution at Cys(564) did not reduce MT4-MMP-stimulated cell invasion of Matrigel suggesting that homodimerization is not required for this process. Deglycosylation studies showed that MT4-MMP is modified by N-glycosylation. Moreover, inhibition of N-glycosylation by tunicamycin diminished the extent of MT4-MMP dimerization suggesting that N-glycans may confer stability to the dimeric form. Taken together, the data presented here provide a new insight into the characteristics of MT4-MMP and highlight the common and distinct properties of the glycosylphosphatidylinositol-anchored membrane type-matrix metalloproteinases.     

The effect of some osmolytes on the activity and stability of mushroom tyrosinase

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) fromAgaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40°C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obeye the first order law. Inactivation rate constant (k_inact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of k_inact(0.7×10⁻⁴s⁻¹) in comparison with their absence (2.5×10⁻⁴s⁻¹). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodyanamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.     

化学添加剂提高重组α-环糊精葡萄糖基转移酶酶制剂稳定性

通过向重组α-环糊精葡萄糖基转移酶 (α-CGT酶) 液中添加化学添加剂以提高其热稳定性及贮存稳定性。在不同温度下研究了添加剂对酶液的贮存稳定性影响,并用圆二色谱 (CD) 研究了CGT酶在近紫外区和远紫外区蛋白质结构与热稳定性的变化关系。当单独加入各种添加剂在50 ℃水浴1 h和室温放置108 d后,发现含有20％甘油的酶液稳定性最好,与未加任何添加剂的对照酶液相比仍有91％和50％的酶活,对照酶液在50 ℃水浴1 h后仅有小于10％的活性,室温放置108 d后已经没有酶活。明胶、CaCl2和PEG40     

Influence of chloride ligands on the structure of Zn- and Cd-metallothionein species

It is now commonly accepted that non-proteic ligands contribute to the structure and stability of metal-metallothionein (M-MT) species, although this contribution may differ substantially depending on the MT and the metal ions involved. Conversely, literature data are unconnected, lacking correlation studies between the contribution of inorganic ligands to the M-MT complexes and the corresponding CD and UV-vis fingerprints. To contribute towards filling this gap, we have analyzed the influence of chloride anions in the Zn- and Cd-MT complexes of mammalian MT1 and MT4 isoforms. Starting from the initial hypothesis that the shoulders appearing at 240nm in the UV-vis difference spectra during the Cd(II) titrations of Zn-MTs would be indicative of chloride participation in these metal-MT complexes, we can now propose that, while their absence definitely rules out these ligands being involved in metal coordination, their presence should not necessarily be attributed to the formation of metal-Cl bonds. Instead, we identified a global blue shift for the UV-vis difference spectral envelope as the most liable indication of chloride participation in the binding sites of the M-MT species. Following this criterion, we determined that chloride anions are bound to the Cd(7)-MT1 and Cd(4)-alphaMT1 complexes but not in the isostoichiometric Zn complexes, nor in the Zn- or Cd-complexes of the homologous MT4 peptides.     

Characterization of alpha-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

alpha-mannosidase from Erythrina indica seeds is a Zn(2+) dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 degrees C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol(-1). N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3-8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of alpha-helical structure in the enzyme. alpha-Mannosidase from E indica exhibits immunological identity with alpha-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with alpha-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of alpha-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate

Paramecium bursaria chlorella virus, a large DNA virus that replicates in unicellular Chlorella-like algae, encodes an RNA triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5' end of the viral mRNAs. The Chlorella virus RNA triphosphatase is the smallest member of the metal-dependent RNA triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and CD studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent non-competitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both CD and guanidinium chloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture was occurring upon decavanadate binding. However, both fluorescence spectroscopy and CD experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA triphosphatases by decavanadate.     

CD44 directs membrane-type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin-like domain

Membrane-type 1 matrix metalloproteinase (MT1- MMP) localizes at the front of migrating cells and degrades the extracellular matrix barrier during cancer invasion. However, it is poorly understood how the polarized distribution of MT1-MMP at the migration front is regulated. Here, we demonstrate that MT1-MMP forms a complex with CD44H via the hemopexin-like (PEX) domain. A mutant MT1-MMP lacking the PEX domain failed to bind CD44H and did not localize at the lamellipodia. The cytoplasmic tail of CD44H, which comprises interfaces that associate with the actin cytoskeleton, was important for its localization at lamellipodia. Overexpression of a CD44H mutant lacking the cytoplasmic tail also prevented MT1-MMP from localizing at the lamellipodia. Modulation of F-actin with cytochalasin D revealed that both CD44H and MT1-MMP co-localize closely with the actin cytoskeleton, dependent on the cytoplasmic tail of CD44H. Thus, CD44H appears to act as a linker that connects MT1-MMP to the actin cytoskeleton and to play a role in directing MT1-MMP to the migration front. The PEX domain of MT1-MMP was indispensable in promoting cell migration and CD44H shedding.     

Multiple effects of chemical reagent on enzyme: o-phthalaldehyde-induced inactivation,dissociation and partial unfolding of lactate dehydrogenase from pig heart

The effects of o-phthalaldehyde (OPTA) on lactate dehydrogenase (LDH) have been studied by following changes in enzymatic activity, aggregation state and conformation. Treatment with OPTA resulted in pseudo first-order inactivation of LDH over a wide concentration range of the inhibitor, and the second-order rate constant was estimated to be 1.52 M⁻¹ s⁻¹. The loss of enzyme activity was concomitant with the increases in absorbance at 337 nm and fluorescence intensity at 405 nm. Complete loss of enzyme activity was accompanied by the formation of approximately 4 mol isoindole derivatives per mole LDH subunit. Cross-linking experiments verified enzyme dissociation during OPTA modification, which could be attributed to the modification of both thiol groups and lysine residues. Circular dichroism (CD) spectra showed that the secondary structure of the OPTA-modified enzyme decreased correspondingly. Comparison of the inactivation with the conformational changes of the enzyme suggests that the active site of the enzyme exhibits greater conformational flexibility than the enzyme molecule as a whole. It is concluded that OPTA modification has multiple effects on LDH, including its inactivation, dissociation and partial unfolding.     

The effect of modification of lactate and malate dehydrogenases on their stability and activity

The inactivation of lactate and malate dehydrogenases (LDH and MDH) modified by progesterone in the water-dimethylformamide (DMF) medium is described by the first-order equation up to large conversion degrees. The MDH modification is accompanied by the increase of its stability by 7-14%, while LDH modification leads to the enzyme stability decrease by 67%. The enzymes catalytic activities are changed simultaneously. The main factors of the stability and activity changes are the DMF influence upon the quaternary structure of the proteins at the modification and a hydrofobization of the external and internal protein sites by progesterone.     

Successful resonance Raman study of cresolase activity of mushroom tyrosinase

Mono-oxygenase (cresolase) activity of mushroom tyrosinase (MT) in the presence of 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide (HPABS) was successfully studied by resonance Raman (rR) spectroscopy. HPABS is a synthetic competitive inhibitor (K(i)=7.17 x 10(-6)M) for the cresolase activity with a large extinction coefficient at 365 nm. Upon reacting with MT, HPABS produced an enzyme-inhibitor (EI) complex with sufficiently long life span. Analyzing the ensuing spectrum indicates that the azo tautomer of HPABS binds to the enzyme and retains its geometrical isomeric form in the EI complex. The observed changes in the rR spectrum of HPABS after binding to MT support the idea that an electrophilic attack on the inhibitor has happened. Similar experiments were designed for studying the oxidase activity of MT. However, the enzymatic reaction, even in the presence of 4-[(2,4-dinitrophenyl)azo]-1,2-benzenediols was still fast enough to tan the reaction solution quickly and render its rR spectrum impregnable background.     

The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea.

Shikimate kinase was chosen as a convenient representative example of the subclass of alpha/beta proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 m urea. From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJ.mol-1. Approximately 95% of the enzyme activity could be recovered on dilution of the urea from 4 to 0.36 m. The results of spectroscopic studies indicated that refolding occurred in at least four kinetic phases, the slowest of which (k = 0.009 s-1) corresponded with the regain of shikimate binding and of enzyme activity. The two most rapid phases were associated with a substantial increase in the binding of 8-anilino-1-naphthalenesulfonic acid with only modest changes in the far UV CD, indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly. The relevance of the results to the folding of other alpha/beta domain proteins is discussed.     

Fish and molluscan metallothioneins

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.     

Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration

Migratory cells including invasive tumor cells frequently express CD44, a major receptor for hyaluronan and membrane-type 1 matrix metalloproteinase (MT1-MMP) that degrades extracellular matrix at the pericellular region. In this study, we demonstrate that MT1-MMP acts as a processing enzyme for CD44H, releasing it into the medium as a soluble 70-kD fragment. Furthermore, this processing event stimulates cell motility; however, expression of either CD44H or MT1-MMP alone did not stimulate cell motility. Coexpression of MT1-MMP and mutant CD44H lacking the MT1-MMP-processing site did not result in shedding and did not promote cell migration, suggesting that the processing of CD44H by MT1-MMP is critical in the migratory stimulation. Moreover, expression of the mutant CD44H inhibited the cell migration promoted by CD44H and MT1-MMP in a dominant-negative manner. The pancreatic tumor cell line, MIA PaCa-2, was found to shed the 70-kD CD44H fragment in a MT1-MMP-dependent manner. Expression of the mutant CD44H in the cells as well as MMP inhibitor treatment effectively inhibited the migration, suggesting that MIA PaCa-2 cells indeed use the CD44H and MT1-MMP as migratory devices. These findings revealed a novel interaction of the two molecules that have each been implicated in tumor cell migration and invasion.     

Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris

A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).     

Horseradish peroxidase thermostabilization: The combinatorial effects of the surface modification and the polyols

Horseradish peroxidase is an important heme-containing plant enzyme with enormous medical diagnostic, biosensing, bioremediation and biotechnological applications. Any improvement in the stability of the enzyme will greatly enhance its application in mentioned areas. In the present study, the stabilizing effects of certain additives and chemical modification by citraconic anhydride on the thermal behavior of HRP were investigated. Both strategies brought about dramatic enhancement of the thermostability of the enzyme. Results obtained on T_m, changes in the circular dichroism (CD) spectra and kinetic parameters of HRP and its modified form are discussed in terms of contributions to the mechanism of the thermal stability and the activity enhancement. Polyols were very effective in providing protection against the irreversible thermoinactivation of the native and the modified forms of the enzyme. Our results reveal that a combination of medium change and surface modification may provide an effective strategy for the enhancement of the thermodynamic and the kinetic stability of the enzyme.     

Structural properties of arrestin studied by chemical modification and circular dichroism.

A unique conformation of arrestin is crucial for its interaction with phosphorylated photolyzed rhodopsin. Conformational changes in arrestin were investigated using chemical modification and circular dichroism. We studied the kinetics of sulfhydryl modification of bovine arrestin in order to determine whether its conformation is altered by the presence of ligands or salts at different ionic strengths. We found that all three cysteines (stoichiometry was 2.7 +/- 0.06 3-carboxy-4-nitrophenyl sulfide (NbS)/arrestin) are accessible for modification by NbS2. Under pseudo-first-order conditions (30-100-fold excess of NbS2 over arrestin), the modifications of the 3 cysteines are indistinguishable. At higher concentrations of NbS2 (150-300-fold excess), the pseudo-first-order plot is not linear, and the reaction can be resolved into two processes that involve two classes of sulfhydryl groups. Addition of CaCl2, MgCl2, inorganic phosphate, MgATP, or MgGTP had little effect on the rate of modification of the cysteine residues; however, heparin and inositol hexakisphosphate, which have been shown to induce conformational changes in arrestin, block modification of one sulfhydryl group of arrestin and accelerate the modification of the remaining two. Analysis of CD spectra revealed that arrestin has virtually no alpha-helical structure, about 40% beta-structure, about 18% beta-turns, and about 40% other structure. The CD spectrum for arrestin did not change in the presence of heparin. These studies suggest that arrestin exists in equilibrium between two or more conformational states. However, it is proposed that conversion between these conformations occur without altering significantly the secondary structure of arrestin.     

Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase]

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.